Pantanella Fabrizio, Valenti Piera, Frioni Alessandra, Natalizi Tiziana, Coltella Luana, Berlutti Francesca
Department of Public Health Sciences, Sapienza University of Rome, Piazzale A. Moro 5, Rome, Italy.
J Microbiol Methods. 2008 Dec;75(3):478-84. doi: 10.1016/j.mimet.2008.07.027. Epub 2008 Jul 30.
The medical device-related infections are frequently a consequence of Staphylococcus biofilm, a lifestyle enhancing bacterial resistance to antibiotics. Antibiotic susceptibility tests are usually performed on planktonic forms of clinical isolates. Some methods have been developed to perform antibiotic susceptibility tests on biofilm. However, none of them counts bacterial inoculum. As antibiotic susceptibility is related to bacterial inoculum, the test results could be mistaken. Here, a new method, BioTimer Assay (BTA), able to count bacteria in biofilm without any manipulation of samples, is presented. Moreover, the BTA method is applied to analyze antibiotic susceptibility of six Staphylococcus strains in biofilm and to determine the number of viable bacteria in the presence of sub-inhibitory doses of four different antibiotics. To validate BTA, the new method was compared to reference methods both for counting and antibiotic susceptibility tests. A high agreement between BTA and reference methods is found on planktonic forms. Therefore, BTA was employed to count bacteria in biofilm and to analyze biofilm antibiotic susceptibility. Results confirm the high resistance to antibiotics of Staphylococcus biofilm. Moreover, BTA counts the number of viable bacteria in the presence of sub-inhibitory doses of antibiotics. The results show that the number of viable bacteria depends on sub-inhibitory doses, age of biofilm and type of antibiotic. In particular, differently to gentamicin and ampicillin, sub-inhibitory doses of ofloxacin and azithromycin reduce the number of viable bacteria at lower extent in young than in old biofilm. In conclusion, BTA is a reliable, rapid, easy-to-perform, and versatile method, and it can be considered a useful tool to analyze antibiotic susceptibility of Staphylococcus spp. in biofilm.
与医疗设备相关的感染通常是由葡萄球菌生物膜引起的，这种生存方式增强了细菌对抗生素的耐药性。抗生素敏感性测试通常针对临床分离株的浮游形式进行。已经开发了一些方法来对生物膜进行抗生素敏感性测试。然而，这些方法都没有对细菌接种量进行计数。由于抗生素敏感性与细菌接种量有关，测试结果可能会出现错误。在此，提出了一种新方法——生物定时器测定法（BTA），该方法能够在不对样品进行任何处理的情况下对生物膜中的细菌进行计数。此外，BTA方法被用于分析六种葡萄球菌菌株在生物膜中的抗生素敏感性，并确定在四种不同抗生素亚抑菌剂量存在下的活菌数量。为了验证BTA，将这种新方法与参考方法在计数和抗生素敏感性测试方面进行了比较。在浮游形式上，发现BTA与参考方法之间具有高度一致性。因此，BTA被用于对生物膜中的细菌进行计数并分析生物膜的抗生素敏感性。结果证实了葡萄球菌生物膜对抗生素具有高度耐药性。此外，BTA能够在抗生素亚抑菌剂量存在的情况下对活菌数量进行计数。结果表明，活菌数量取决于亚抑菌剂量、生物膜的年龄和抗生素的类型。特别是，与庆大霉素和氨苄西林不同，亚抑菌剂量的氧氟沙星和阿奇霉素在年轻生物膜中比在老年生物膜中降低活菌数量的程度要小。总之，BTA是一种可靠、快速、易于操作且通用的方法，可被视为分析生物膜中葡萄球菌属抗生素敏感性的有用工具。
